This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. (A) OBJECTIVES The architecture of the transverse tubular system (t-system) and the arrangement of associated proteins are central to the function of ventricular cardiomyocytes. The objective of our research is to study structure-function relationships of the t-system and its associated proteins. In our experimental studies, we apply confocal imaging of ventricular myocytes and methods of digital image processing to characterize the geometry of the t-system in rabbit and human cells. This allows us to quantify the distribution of t-system associated proteins such as ryanodine receptors (RyRs), neuronal (brain-type) sodium channels, and sodium-calcium exchangers (NCX), and to study the involvement of these t-system associated proteins in excitation-contraction (EC) coupling. Some of the issues we want to investigate with our experimental studies are the effects of t-tubular geometry on the diffusion of ions and nutrients in the t-system as well as the contribution of NCX and neuronal sodium channels to EC coupling. To test our experimental hypotheses we will use the NBCR tools and algorithms for the computational simulations of biomedical systems designed, developed and implemented by Dr. Holst and colleagues. Our specific aims in the proposed computational studies are: Specific Aim 1: to characterize the effects of t-tubular constrictions on calcium signaling in rabbit ventricular myocytes. In previous work, we found that constrictions occur with a spacing of 1.87[unreadable]1.09 mm along t-tubules (Savio-Galimberti et al., 2008). We speculated that this local variation in t-tubular cross-sectional area may contribute to significant local inhomogeneities of calcium and sodium concentrations in the t-system. With the UCSD sub- cellular modeling environment, we will simulate these local inhomogeneities of ionic concentrations and test our hypotheses that these inhomogeneities affect EC coupling. Specific Aim 2: to determine whether mechanical straining of t-tubules can contribute to ion transport into and out of the t-system. In previous work, we showed that t-tubules exhibit significant flattening. The ratio of the major to the minor diameters of t-tubular cross- sections was 0.73[unreadable]0.14 (Savio et al., 2007). The minor axes of t-tubular cross-sections were approximately parallel to the long axis of myocytes. The flattening of t-tubules may be related to the cells being at slack length. If this is the case it suggests that when cells shorten or lengthen a local volume change within the t-system could take place. Currently, it is unknown if and to what extent such a volume change can contribute to ion transport, which is currently thought to be only by diffusion. The UCSD sub-cellular modeling environment is an ideal platform to characterize the effects of these volume changes. Specific Aim 3: to use the UCSD modeling environment to study the effects of sodium fluxes on EC coupling. Our experimental hypotheses are that sodium flux through neuronal sodium channels can influence EC coupling presumably by reversing NCX. Reverse NCX could have a significant effect on EC coupling by priming the dyadic cleft with calcium (Sobie et al., 2008) and increasing the gain for triggered sarcoplasmic calcium release (i.e. the ratio of calcium release flux to trigger flux). An important question is how these sodium fluxes affect EC coupling.